Erythrocyte protein 4.1 (4.1R) is an 80 kD cytoskeletal protein that stabilizes the spectrin/actin skeleton and attaches it to the membrane. This form is only one of a complex family of isoforms arising from a single 4.1R gene by alternative mRNA splicing and posttranslational modification. Antibodies specific to 4.1R react with cell nuclei, centrosomes, and parts of the mitotic apparatus in dividing cells as well as the cell-cell junctions of epithelial cells. Our studies of 4.1R in nucleated mammalian cells have shown that at least one 135 kD 4.1R isoform specifically interacts with Nuclear-Mitotic Apparatus protein (NuMA) and two isoforms (135 kD and 150 kD) interact with the tight junction protein Zonula occludens (ZO)-2. These isoforms associate with complexes containing proteins involved in mitosis and tight junctions, respectively. Overexpressing a 135 kD isoform disrupts the nuclear localization of NuMA and reduces survival of transfectants. Overexpression of the ZO-2 interacting domain of 4.1 R decreases the transepithelial resistance of the transfected cells. 4. 1 R expression is also suppressed in neuroblastoma cells. Our findings support the hypothesis that some non-erythroid 4.1R species play pivotal roles in nuclear architecture, cell proliferation and cell maturation processes, tight junction biogenesis, and, possibly, tumor suppression. We now propose to test these hypotheses by pursuing studies designed to examine the role of 4.1R isoforms during cell division, epithelial maturation, and tight junction formation. We will also determine whether 4.1R plays a role in tumor suppression in both cultured cell models and primary-cells derived from 4.1R+/+ and -/- knock out mice. We shall identify the 4.1R isoforms involved in these processes at both the mRNA and protein level. The physiological importance of 4.1R in cell division and tight junction biogenesis and epithelial maturation will be assessed by reconstitution of 4. 1 R function in the -/- cells and by dominant negative mutagenesis. The role of 4.1R in cell cycle arrest, apoptosis, and genetic stability will also be analyzed using established in vitro and in vivo approaches. We expect our studies to clarify the role of this cytoskeletal protein in these critical physiological processes.